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• Transcription factors (TFs) may play a central role in plant morphological evolution. Variation in the nonsynonymous to synonymous nucleotide substitution rate (dN/dS) ratio among TFs can be attributed to either differences in constraint or the frequency of adaptive substitution. However, the relative contribution of these forces to the variation in dN/dS ratios is unknown. • We synthesize current and previous results comparing the variation in dN/dS ratios among members of the MYB-bHLH-WDR complex of TFs that regulates floral anthocyanin pigmentation in Ipomoea. • Low values of dN/dS in a WDR gene are the result of exceptionally strong purifying selection, with no evidence of positive selection. bHLH and MYB genes also fail to show evidence for positive selection, but have higher dN/dS ratios, indicating reduced selective constraint. • Differences in constraint are consistent with expectations based on the intrinsic features and regulatory network properties among these proteins. Significantly elevated dN/dS ratios in the MYB gene suggest that mutations experience reduced magnitudes of deleterious pleiotropy compared with the rest of the complex. Although reduced constraint may account for the observation that Myb mutations disproportionately contribute to differences in floral pigmentation, the lack of detectable positive selection in any of these TF proteins suggests that amino acid substitutions contribute little to flower colour evolution., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Stimulation of macrophages with lipopolysaccharide (LPS) leads to the production of cytokines that elicit massive liver apoptosis. We investigated the in vivo role of stress-responsive transcription factors (SRTFs) in this process focusing on the precipitating events that are sensitive to a cell-permeant peptide inhibitor of SRTF nuclear import (cSN50). In the absence of cSN50, mice challenged with LPS displayed very early bursts of inflammatory cytokines/chemokines, tumor necrosis factor alpha (1 h), interleukin 6 (2 h), interleukin 1 beta (2 h), and monocyte chemoattractant protein 1 (2 h). Activation of both initiator caspases 8 and 9 and effector caspase 3 was noted 4 h later when full-blown DNA fragmentation and chromatin condensation were first observed (6 h). At this time an increase of pro-apoptotic Bax gene expression was observed. It was preceded by a decrease of anti-apoptotic Bcl2 and BclX(L) gene transcripts. Massive apoptosis was accompanied by microvascular injury manifested by hemorrhagic necrosis and a precipitous drop in blood platelets observed at 6 h. An increase in fibrinogen/fibrin degradation products and a rise in plasminogen activator inhibitor 1 occurred between 4 and 6 h. Inhibition of SRTFs nuclear import with the cSN50 peptide abrogated all these changes and increased survival from 7 to 71%. Thus, the nuclear import of SRTFs induced by LPS is a prerequisite for activation of the genetic program that governs cytokines/chemokines production, liver apoptosis, microvascular injury, and death. These results should facilitate the rational design of drugs that protect the liver from inflammation-driven apoptosis.

Genetic programming of B lymphocytes plays a key role in development of autoimmune diabetes in NOD mice. The genetic programs for autoimmunity, inflammation and apoptosis are regulated by NF-kappa B and other stress-responsive transcription factors (SRTFs). In response to inflammatory, immune, and oxidant stress, these SRTFs translocate to the nucleus via a set of nuclear import adapters known as karyopherins/importins. Karyopherin alpha 2 (Rch1, KPNA2) is the target for a cell-penetrating peptide (cSN50) that prevents the nuclear import of SRTFs. Here we report that cSN50 prevents overt diabetes in an accelerated model in which 10-11 week old female NOD mice received cyclophosphamide (Cy) (200mg/kg). Following Cy administration, NOD splenocytes displayed increased nuclear localization of NF-kappa B, and NOD B lymphocytes showed upregulation of CD80 and CD86. These preliminary findings indicate that Cy-induced acceleration is consistent with a model of antigen presenting cell/B cell-mediated autoimmunity. We treated Cy-challenged NOD mice with cSN50 for two days beginning 45 hours after Cy. Only 10% of cSN50 treated mice developed hyperglycemia during 250 days of followup compared to 90% of saline- or control peptide-treated animals (p=0.0002, log-rank). After 24 hours of cSN50 therapy, islets from mice receiving cSN50 demonstrated nearly complete resolution of insulitis by immunohistochemistry. Mice treated with cSN50 had reduced B lymphocyte numbers in pancreas, spleen and pancreatic lymph nodes. No changes were noted in the proportion of activated T ceils (CD69+), regulatory T cells (foxp3+), or T cell-derived cytokines. In contrast, cSN50-treated animals demonstrated increased serum levels of IL-6 (p<0.003, ANOVA) and MCP-1 (p<0.02) during therapy, factors that may be islet protective. These cytokines/chemokines were B cell-derived as restimulation of splenocytes from cSN50-treated mice with B cell agonists but not T cell agonists recapitulated this cytokine/chemokine profile. Cumulatively, these results provide initial evidence that re-programming of B lymphocytes by nuclear import modulation suppresses autoimmune diabetes after the establishment of insulitis. [ABSTRACT FROM AUTHOR]